An Abundant Tissue Macrophage Population in the Adult Murine Heart with a Distinct Alternatively-Activated Macrophage Profile

Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP+ population within the adult Cx3cr1GFP/+ knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45+CD11b+GFP+) are distinct from mononuclear CD45+CD11b+GFP+ cells sorted from the spleen and brain of adult Cx3cr1GFP/+ mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis

Multiple congenital malformations of Wolf-Hirschhorn syndrome are recapitulated in Fgfrl1 null mice

Wolf-Hirschhorn syndrome (WHS) is caused by deletions in the short arm of chromosome 4 (4p) and occurs in about one per 20,000 births. Patients with WHS display a set of highly variable characteristics including craniofacial dysgenesis, mental retardation, speech problems, congenital heart defects, short stature and a variety of skeletal anomalies. Analysis of patients with 4p deletions has identified two WHS critical regions (WHSCRs); however, deletions targeting mouse WHSCRs do not recapitulate the classical WHS defects, and the genes contributing to WHS have not been conclusively established. Recently, the human FGFRL1 gene, encoding a putative fibroblast growth factor (FGF) decoy receptor, has been implicated in the craniofacial phenotype of a WHS patient. Here, we report that targeted deletion of the mouse Fgfrl1 gene recapitulates a broad array of WHS phenotypes, including abnormal craniofacial development, axial and appendicular skeletal anomalies, and congenital heart defects. Fgfrl1 null mutants also display a transient foetal anaemia and a fully penetrant diaphragm defect, causing prenatal and perinatal lethality. Together, these data support a wider role for Fgfrl1 in development, implicate FGFRL1 insufficiency in WHS, and provide a novel animal model to dissect the complex aetiology of this human disease

Proximal tubule overexpression of a locally acting IGF isoform, Igf-1Ea, increases inflammation after ischemic injury

Objective: IGF-1 is an important regulator of postnatal growth in mammals. In mice, a non-circulating, locally acting isoform of IGF-1, IGF-1Ea, has been documented as a central regulator of muscle regeneration and has been shown to improve repair in the heart and skin. In this study, we examine whether local production of IGF1-Ea protein improves tubular repair after renal ischemia reperfusion injury

Overexpression of mIGF-1 in keratinocytes improves wound healing and accelerates hair follicle formation and cycling in mice

Insulin-like growth factor 1 (IGF-1) is an important regulator of growth, survival, and differentiation in many tissues. It is produced in several isoforms that differ in their N-terminal signal peptide and C-terminal extension peptide. The locally acting isoform of IGF-1 (mIGF-1) was previously shown to enhance the regeneration of both muscle and heart. In this study, we tested the therapeutic potential of mIGF-1 in the skin by generating a transgenic mouse model in which mIGF-1 expression is driven by the keratin 14 promoter. IGF-1 levels were unchanged in the sera of hemizygous K14/mIGF-1 transgenic animals whose growth was unaffected. A skin analysis of young animals revealed normal architecture and thickness as well as proper expression of differentiation and proliferation markers. No malignant tumors were formed. Normal homeostasis of the putative stem cell compartment was also maintained. Healing of full-thickness excisional wounds was accelerated because of increased proliferation and migration of keratinocytes, whereas inflammation, granulation tissue formation, and scarring were not obviously affected. In addition, mIGF-1 promoted late hair follicle morphogenesis and cycling. To our knowledge, this is the first work to characterize the simultaneous, stimulatory effect of IGF-1 delivery to keratinocytes on two types of regeneration processes within a single mouse model. Our analysis supports the use of mIGF-1 for skin and hair regeneration and describes a potential cell type-restricted action

Immunophenotype of cTMs within adult <i>Cx₃cr1^GFP/+</i> mouse hearts.

<p>Representative histograms for myeloid (F4/80, CD11c, MHC-II (IAb), CD14, CD86) and lymphoid (CD3ε, B220) related markers detected in CD45⁺CD11b⁺GFP⁺ population and CD45⁺CD11b⁻GFP⁻ population. Red line shows isotype control staining. Histograms representative of at least 4 experiments, with mouse group sizes of at least 4 mice.</p

Differential gene expression by GFP⁺ populations from adult <i>Cx₃cr1^GFP/+</i> mouse tissues determined by qRT-PCR.

<p>Means for respective genes from various tissues are normalised to the means obtain for heart (IL1β, IL6, CXCL1, CXCL2, IL10, IGF1, MMP13, Timp2, and Hmox1), lung (Angpt1), or peritoneum (PT; Arg1). Histograms show mean ± SEM (n = 3 independent cell and RNA isolation experiments). RNA for 3 biological replicate samples of GFP⁺ cells of various tissues were prepared in 3 independent isolation procedures for each tissue. For all tissues except spleen, GFP⁺ cells were isolated from 4–7 mice in each independent cell isolation. Splenic GFP⁺ cells were isolated from groups of 2–4 mice per replicate.</p

Expression of complement opsonisation components cTMs and GFP⁺ cells from the spleen and brain.

<p>Microarray analysis derived heat maps of complement pathway related gene expression by cTMs and GFP⁺ cells from the spleen and brain identified by microarray gene expression analysis. (A) Secreted complement factors with probes corresponding to C1q members highlighted. (B) Complement receptors.</p

Detection of GFP⁺ cells within adult <i>Cx₃cr1^GFP/+</i> mouse hearts.

<p>(A) Scatter profiles showing CD45⁺ cells in the adult mouse heart (left panel) and the expression of CD11b and GFP within this population (right panel). (B) Scatter profile of GFP⁺ cells (green), with CD45⁺GFP⁻ (black) and CD45⁻GFP⁻ cells and debris (grey) shown (top left panel). (Top right panel) scatter profile of CD45⁺ cells (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036814#pone-0036814-g001" target="_blank">figure 1A left panel</a>) with GFP⁺ cells, GFP⁻ cells and debris indicated (green, black and grey respectively; based on gating from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036814#pone-0036814-g001" target="_blank">figure 1A</a>). (Bottom left panel) scatter profile of CD11b and CD45 expression within GFP⁺ cells. (C) 2 MDa dextran uptake by CD45⁺CD11b⁺GFP⁺ cells. (D) 45 µm projection view of a cross sectional image of the adult <i>Cx₃cr1^GFP/+</i> mouse heart left ventricle. Scale bar = 150 µm. (E) 3D projections of typical GFP⁺ (green) cells from the left ventricular myocardium with isosurface rendering of nuclei (blue). Figures representative of at least 3 independent experiments. (F) Magnified projection view of GFP⁺ cells with perivascular position shown (capillaries stained with IB4; nuclei stained with DAPI). Scale bar = 5 µm. (G) Optical section of GFP+ cells with perivascular (capillaries stained with IB4) and pericardiomyocyte position (cardiomyocyte cell surface stained with WGA; nuclei stained with DAPI). Scale bar = 10 µm. All images representative of at least 3 similar independent experiments.</p

Expression of M2 macrophage markers by cTMs.

<p>(A) Heat map of M2 related gene expression by cTMs and GFP⁺ cells from the spleen and brain by microarray analysis. (B) Scatter/correlation profile of M2 related genes expressed by cTMs (heart) and genes expressed in GFP⁺ cells from the spleen and brain. (C) Micrographs of adult <i>Cx₃cr1^GFP/+</i> mouse heart sections stained for GFP, Mrc1, CD163 and Lyve-1. Scale bar = 30 µm. (D) Flow cytometry histogram showing expression of M2 markers Mrc1 and Ly6C/G by cTMs. Histograms are representative of at least 4 independent experiments. (E) qRT-PCR analysis of Lyve-1 expression by GFP⁺ cells from different tissues. Mean values determined for respective tissues are normalised to means obtained for heart. Histograms show mean ± SEM (n = 3).</p

Microarray gene expression analysis of cTMs and GFP⁺ cells from the spleen and brain.

<p>(A) Venn-diagram of differentially expressed genes from cTMs (blue) and GFP+ cells from the spleen (green) and brain (red). (B) Heat map of genes enriched greater than 20-fold in cTMs compared to GFP⁺ cells from the spleen and brain. Heat map displays data from each individually isolated GFP⁺ populations. (C) qRT-PCR histograms conducted on selected genes to determine unique enrichment in cTMs (Lyve-1, IGF1, MMP13, IL10, IL6, IL1b, CXCL1, and CXCl2), enrichment in both cTMs and GFP+ cells from the brain (Stab1, Timp2), enrichment in GFP⁺ cells from the spleen (Angpt1) and expression in all three populations (Hmox1). Histograms show mean ± SEM. For microarray analysis, 3 biological replicate samples of GFP⁺ cells from the heart, spleen and brain were prepared in 3 independent isolation procedures for each tissue. Cardiac and brain tissue from 4 mice were used to isolate sufficient number of cells in each independent cell isolation, whereas splenic cells were isolated from at least 2–4 mice.</p